1a.Objectives (from AD-416)
The long-term objective of this project is to develop improved rice (Oryza sativa L.) germplasm for use in breeding elite varieties adapted to temperate environments by identifying, characterizing, and manipulating genes that affect crop productivity. In temperate regions, seedling cold tolerance in rice is important for successful stand establishment and plant development, both of which directly impact yield. Over the next five years, two major objectives will be addressed:.1)the molecular genetic basis of rice seedling cold tolerance conferred by the major quantitative trait loci qCTS12 and qCTS4 will be determined and.2)the information obtained from the analysis of qCTS12 and qCTS4 will be used to evaluate currently available germplasm and to develop new germplasm with enhanced cold tolerance. qCTS12 and qCTS4 have been fine mapped to regions containing a small number of genes.

1b.Approach (from AD-416)
During this project, the genes that confer cold tolerance will be identified through transformation and candidate gene analysis experiments. The genes and the proteins they encode will be characterized at the molecular level and their effect under field conditions will be determined. The utility of this of this information for evaluating other rice germplasm and for developing efficient approaches to identifying novel sources of cold tolerance will be examined. New germplasm for breeding will be developed by transferring the qCTS12 and qCTS4 genes through conventional and molecular breeding approaches. In addition to providing tools and resources for germplasm improvement, genetic dissection of qCTS12 and qCTS4 will contribute to our fundamental understanding of cold tolerance in rice. Formerly 5306-21000-016-00D (3/08).

3.Progress Report
Functional analysis of qCTS12 candidate gene: Progress was made on identifying the gene underlying the seedling cold tolerance QTL designated qCTS12. Previously, the gene OsGSTZ2, which encodes a glutathione S-transferase enzyme of the zeta class (GSTZ), was found to have 2-3X greater expression in tolerant (cv. M-202) compared to sensitive rice (cv. IR50) under low temperatures. Two amino acid differences resulting from single nucleotide polymorphisms between the M-202 and IR50 alleles were identified and shown to be correlated with visual assessment of seedling cold tolerance in a small set (n = 20) of rice lines. GSTZ (also known as maleylacetoacetate isomerase; MAAI) has little or no activity on traditional GST substrates, but instead exhibits an isomerase activity that converts maleylacetone to fumarylacetone. This conversion represents the penultimate step in the tyrosine and phenylalanine catabolic pathway. In FY10, recombinant GSTZ2 proteins from M-202 and IR50 genes were produced and purified to examine the activity of the enzymes. In addition, constructs containing each individual amino acid difference were made, expressed, and purified. Using standard GST substrates (i.e., 1-chloro-2,4-dinitrobenzene, ethacrynic acid, benzyl isothiocyanate), little activity was observed with the GSTZ2 proteins. Using a simple assay based on GSTZ’s dechlorinating activity toward dichloroacetic acid, we observed that the M-202 GSTZ2 protein exhibited strong activity while the IR50 enzyme did not. Furthermore, this difference in activity was due to the difference of a single amino acid at position 99 (isoleucine in M-202, valine in IR50). The more complex analysis of MAAI activity of the recombinant proteins is in progress.

Characterization of rice seedling response to low temperature stress: Identification of qCTS12 and qCTS4 loci was based on visual ratings of rice seedlings. Such ratings have limitations with regard to screening germplasm for other sources of tolerance. To determine if more quantitative assays may be used to detect qCTS12, qCTS4 and other important cold tolerance loci, the response of M-202 and IR50 to low temperature stress was assessed by examining:.1)electrolyte leakage;.2)chlorophyll/anthocyanin content;.3)proline content;.4)lipid oxidation; and.5)reduced ascorbic acid and glutathione content. Under constant low temperature stress (i.e., qCTS12 conditions), M-202 and IR50 were clearly distinguished using electrolyte leakage and proline content. Effects on chlorophyll/anthocyanin, lipids, ascorbic acid and glutathione were observed after longer exposure. Results indicate that timing of the assays needs to be refined. Interestingly, although comparison of electrolyte leakage and visual rating of M-202 and IR50 were consistent, preliminary analyses of other germplasm were not in agreement (i.e., some germplasm visually rated as cold sensitive had relatively low electrolyte leakage). This may indicate the involvement of other genes. In addition to refining these assays, we will begin employing them to assess the response of M-202 and IR50 to intermittent stress (i.e., qCTS4 conditions).

4.Accomplishments
1.
Analysis of a candidate gene for rice seedling cold tolerance. Exposure of rice seedlings to low temperatures can result in stunting or death and ultimately a reduction in yield and grain quality. ARS Scientists in Davis, CA have identified a gene called OsGSTZ2, which appears to contribute to seedling cold tolerance, and have undertaken studies to confirm its function. This gene encodes an enzyme called glutathione S-transferase zeta. We have shown that the enzymes from cold tolerant and sensitive rice varieties differ in their activity and this may be the reason for the difference in varietal response to low temperatures. Confirming the identification of this seedling cold tolerance gene and determining how it functions will facilitate the breeding of this trait in desirable, but currently cold sensitive varieties.